CA1336585C - Method of stabilizing urokinase precursor and dry preparation containing said precursor - Google Patents
Method of stabilizing urokinase precursor and dry preparation containing said precursorInfo
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- CA1336585C CA1336585C CA000506647A CA506647A CA1336585C CA 1336585 C CA1336585 C CA 1336585C CA 000506647 A CA000506647 A CA 000506647A CA 506647 A CA506647 A CA 506647A CA 1336585 C CA1336585 C CA 1336585C
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- acid
- salts
- precursor
- albumin
- urokinase precursor
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/46—Hydrolases (3)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
- C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
- C12N9/6424—Serine endopeptidases (3.4.21)
- C12N9/6456—Plasminogen activators
- C12N9/6462—Plasminogen activators u-Plasminogen activator (3.4.21.73), i.e. urokinase
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/46—Hydrolases (3)
- A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
- A61K38/49—Urokinase; Tissue plasminogen activator
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/02—Inorganic compounds
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
- A61K47/12—Carboxylic acids; Salts or anhydrides thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
- A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
- A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H5/00—Compounds containing saccharide radicals in which the hetero bonds to oxygen have been replaced by the same number of hetero bonds to halogen, nitrogen, sulfur, selenium, or tellurium
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/21—Serine endopeptidases (3.4.21)
- C12Y304/21073—Serine endopeptidases (3.4.21) u-Plasminogen activator (3.4.21.73), i.e. urokinase
Abstract
A method of stabilizing a urokinase precursor and a dry preparation containing said stabilized urokinase are disclosed. This method comprises adding, as stabilizers, (a) at least one member of inorganic salts, organic acid salts, and polar amino acids and the salts thereof, and (b) albumin to the urokianse precursor. The urokinase precursor-containing preparation is highly stable and can be stored for a long period of time.
Description
METHOD OF STABILIZING UROKINASE PRECURSOR AND
DRY PREPARATION CONTAINING SAID PRECURSOR
The present invention relates to a method of stabilizing a urokinase precursor and a dry preparation containing said precursor.
A urokinase precursor secreted from human kidney cells does not exhibit urokinase activity by itself.
However, when digested with a proteinase, such as plasmin, the precursor is transformed into a molecule which shows a high urokinase activity.
The urokinase precursor has a high affinity to fibrin and reaches the fibrin constituting a blood clot without acting on or decomposing fibrinogen in the blood plasma. Thereafter, the precursor exhibits the urokinase activity by the action of plasmin. That is, the urokinase precursor causes limited fibrin dissolution in the blood clot site and dissolves the blood clot selectively and efficiently. Therefore, the precursor is believed to be useful as a novel medicine for treatment of the disease of thrombosis.
The urokinase precursor is unstable in a highly purified condition, or dried or freeze dried condition, although it is relatively stable when stored in a neutral solvent in a high concentration during the processes of preliminary purification and final purification.
DRY PREPARATION CONTAINING SAID PRECURSOR
The present invention relates to a method of stabilizing a urokinase precursor and a dry preparation containing said precursor.
A urokinase precursor secreted from human kidney cells does not exhibit urokinase activity by itself.
However, when digested with a proteinase, such as plasmin, the precursor is transformed into a molecule which shows a high urokinase activity.
The urokinase precursor has a high affinity to fibrin and reaches the fibrin constituting a blood clot without acting on or decomposing fibrinogen in the blood plasma. Thereafter, the precursor exhibits the urokinase activity by the action of plasmin. That is, the urokinase precursor causes limited fibrin dissolution in the blood clot site and dissolves the blood clot selectively and efficiently. Therefore, the precursor is believed to be useful as a novel medicine for treatment of the disease of thrombosis.
The urokinase precursor is unstable in a highly purified condition, or dried or freeze dried condition, although it is relatively stable when stored in a neutral solvent in a high concentration during the processes of preliminary purification and final purification.
- 2 - l 3365~5 1 SUMMARy OF THE INVENTION
An object of the present invention is to provide a method of stabilizing a urokinase precursor and also to a dry preparation containing a stabilized urokinase precursor.
It has been found that a dried urokinase precursor is stabilized when a mixture of (a) at least one member of inorganic salts, organic acid salts, and polar amino acids and their salts, and (b) human albumin is added to the precursor.
In one embodiment, the present invention relates to a method of stabilizing a urokinase precursor by adding thereto (a) at least one member of inorganic salts, organic acid salts, and polar amino acids and their salts, and (b) albumin.
In another embodiment, the present invention relates to a dry preparation containing a stabilized urokinase precursor.
DETAILED DESCRIPTION OF THE INVENTION
The urokinase precursor as used herein includes those precursors obtained from urine, human kidney cells, and by genetic engineering (see JP-A-62981/1985 corresponding to Canadian Patent Application Serial 1 3365~5 1 No. 462,860 for example).
The effect of the present invention is particularly remarkable in a highly purified urokinase precursor, such as a urokinase precursor of a concentration that the specific activity is 100,000 IU/mg protein. The symbol "IU" is an abbreviation of an international unit of UK. 1 IU/ml, which is e~uivalent to the UK activity when 1 ml of the precursor is treated with plasmin, means that 1 ml of the precursor, when treated with plasmin, has the same activity as that of 1 IU of UK.
Examples of the inorganic salts that are used in the present invention are alkali metal and alkaline earth metal salts of halogen, and alkali metal and alkaline earth metal salts of phosphoric acid. Preferred are sodium chloride, potassium chloride, sodium phosphate, potassium phosphate, and calcium phosphate.
As the organic acids, aliphatic carboxylic acids which may have a hydroxyl group are preferably used. The number of carboxyl groups is preferably 1 to 3 and the number of hydroxyl groups is preferably 0 to 3. Suitable examples of these organic acids are oxy acids such as citric acid, aliphatic dicarboxylic acids such as oxalic acid, and fatty acids such as acetic acid and mandelic acid.
-4- l 336585 l Examples of the salts of the above organic acids are alkali metal (e.g., sodium and potassium) salts.
Examples of the polar amino acids that are used in the present invention are glutamic acid, aspartic acid, arginine, lysine, and histidine.
Suitable examples of the salts of the above polar amino acids are sodium glutamate and arginine chloride.
Among the above substances which can be used as component (a), organic acid salts and poler amino acids, more particularly arginine and sodium citrate, are preferred.
The albumin as used herein is preferably albumin obtained from human in view of the problem of antigenicity. There are no special limitations to the albumin as long as it is purified for the purpose of use for medical treatment. The purity is preferred to be such that the albumin content as determined by electrophoresis is not less than 80%. Methods of obtaining human albumin include the ethanol fractionation method (see Japanese Patent Publication Nos. 2869/72 and 5297/60), and the method comprising heating in the presence of organic acids (see Japanese Patent Publication Nos. 1604/68 and 40132/76). It is particularly preferred to subject the albumin to heat treatment (preferably at 60C for about 10 hours) for inactivation of hepatitis virus, for example.
-_ 5 _ t 33 65~5 l Albumin is added in an amount corresponding to 10 to 60mg, preferably 25 to 50 mg, and the inorganic salt, organic acid salt, or polar amino acid or its salt is added in an amount corresponding to 2 to 50 mg, preferably 5 to 30 mg, per 10,000 to 250, 000 IU of the urokinase precursor.
The stabilizers of the present invention, i.e., inorganic salts, organic acid salts, polar amino acids or their salts, are added with good results at any stage during the process that the urokinase precursor is placed under conditions where the precursor could be inactivated, such as the purification and storage of the urokinase precursor.
The present invention is described in greater detail with reference to the following examples.
The activity of the urokinase precursor was measured, after activation with plasmin, by the use of a synthetic substrate (Glt-Gly-Arg-MCA).
To a solution of a purified urokinase precursor (specific activity: 135,000 IU/mg protein) in a 0.10 M
phosphate buffer (pH:6.0) as a solvent was added a solution a solution of albumin in the same buffer as above to prepare 25,000 IU/ml of an aqueous urokinase precursor solution containing 20 mg/ml of human albumin. Various additives (inorganic salts, organic acid salts, or polar - 6 ~ t 3 3 6 5 ~ 5 1 amino acids or their salts) were added to the above prepared aqueous urokinase precursor solution so that the total amount of the additives added was 8 mg/ml and then freeze dried. For comparison, the aqueous urokinase precursor solution not containing any additive, and the aqueous urokinase precursor solution with only human albumin added thereto were freeze dried in the same manners as above.
These compositions were measured for the remaining urokinase precursor titer (%) just after its freeze drying and after storage at 50C for 3 months. The results are shown in Table 1.
1 Table 1 Residual Titer (~) After Storage Run Human Additive Just after at 50C for No. Albumin (Total Amount) Freeze Dryinq 3 Months (mg/ml) 1 20 Sodium citrate 99.5 99.8 - (8 mg/ml) 2 " Sodium mandelate 99.0 96~1 (8 mg/ml) 3 " Sodium phosophate 96.5 86.5 (8 mg/ml) 4 " NaCl (Table salt) 98.6 95.2 (8 mg/ml) n Potassium chloride 96.5 86.2 (8 mg/ml) 6 " Aspartic acid 97.5 93.8 (6.4 mg/ml) 7 " Glutamic acid 97.2 94.2 (6.4 mg/ml) 8 " Sodium glutamate 97.6 95.8 (6.4 mg/ml) g n Arginine 99.5 98.8 (6.4 mg/ml) n Lysille (6.4 mg/ml) 11 n His~idine 96.5 - 88.6 (6.4 mg/ml) 12 " - 96.7 -- 60.2 3013 - - 94.0 2.5 Note: Run Nos. 1 - 11: Examples of the present invention Run Nos. 12 and 13: Control Freeze dried preparations were prepared in the same manner as in Experiment 1 containing human albumin, sodium citrate, salt (NaCl), arginine, or sodium glutamate which rates were varied.
The preparations were measured for the residual urokinase precursor titer (%) just after preparation and after storage at 50C for 1 month. The results are shown in Table 2.
Table 2 Formation Residual Titer (%) Additive Just After After Storage Run Human Amount Freezeat 50C for No. Albumin Type (mq/ml) Dryinq1 Month 1 20 Sodium citrate - 77.1 75.2 mg/ml - 2 ~ do 3.2 87.3 86~4 3 ~ do 6.4 99.3 99.2 4 Salt - 76.3 75.2 " do 3.2 84.2 84.1 6 - do 6.4~ 97.8 97.4 7 Arginine - 94.0 75.2 8 ~ do 3.2 99.5 86.4 9 ~ do 6.4 99.5 99.2 . Sodium glutamate - 94.0 75.2 11 i~ do 3.2 97.0 84.1 12 n do 6.4 97.6 97.4 l The titer shown in Tables 1 and 2 is a residual activity against the titer before the above treatment as 100 .
25,000 IU of a purified urokinase precursor (specific activity: 135,000 IU/mg protein), 20 mg of human albumin, and 6.4 mg of sodium citrate were dissolved in 1 ml of a 0.1 M phosphate buffer (pH, 7.0), aseptically filtered, charged in a vial bottle, and then freeze dried to prepare 10 an injection preparation containing 25,000 units of urokinase precursor, 20 mg of human albumin and 6.4 mg of sodium citrate.
A preparation containing 25,000 IU of urokinase l5 precursor, 20mg of human albumin and 6.4 mg if table salt (NaCl) was prepared in the same manner as in Example 1.
So,OoO IU of a purified urokinase precursor (specific activity: 135,000 IU/mg protein), 40 mg of human 20 albumin and 12.8 mg of sodium citrate were processed in the same manner as in example 1 to prepare a preparation containing 50,000 IU of urokinase precursor, 40 mg of human albumin and 12.8 mg of sodium citrate.
- lo - 1 3 3 6 5 8 5 25,000 IU of a purified urokinase precursor (specific activity: 135,000 IU/mg protein), 20 ~ of human albumin, and 6.4 mg of arginine were dissolved in 1 ml of a O.lM phosphate buffer (pH, 7.0), aseptically filtered, charged in a vial bottle, and then freeze dried to prepare a preparation containing 25,000 IU of urokinase precursor, 20 mg Of human albumin and 6.4 mg of arginine.
A preparation containing 25,000 IU of urokinase precursor, 20 mg of human albumin and 6.4 mg of sodium glutamate was prepared in the same manner as in Example 1.
50,000 IU of a purified urokinase precursor (specific activity: 135,000 IU/mg Protein), 40 mg of human albumin and 12.8 mg of arginine were processed in the same manner as in Example 1 to prepare a preparation containing 50,000 IU of urokinase precursor, 40 mg of human albumin and 12.8 mg of arginine.
SYNTHESIS EXAMPLE
Cultivated human kidney cells were grown for 3 days in a 0.1% human serum albumin-added non-serum culture, and the fermentation broth thus obtained was subjected to centrifugal separation. The resulting supernatant was frozen and stored. The pooled supernatant was adjusted to l pH 5.5 and contacted with CM-Sephadex C-50. After washing a column with a 0.16 M phosphate buffer (pH 5.5), the adsorbed urokinase precursor was eluted with a 0.16 M phosphate buffer (pH, 8.5).
Among hybridomas prepared by fusion using polyethylene glycol of spleen cells of BALB/c mouse which had been immunized with the urokinase precursor and mouse myeloma cells, a clone having a high productivity of antibody for the urokinase precursor was selected. From this fermentation broth of the fused cells, an anti-urokinase monoclonal antibody was recovered. This monoclonal antibody was immobilized on BrCN-activated Sepharose 4B (produced by Pharmacia Co., Ltd.) This monoclonal antibody column was equlibrated with a 0.1 M phosphate buffer (pH, 7.0) containing 0.4 M
NaCl, and then contacted with the elute containing urokinase precursor. After washing the column with a 0.1 M phosphate buffer (pH, 7.0) containing 0.4 M NaCl, the adsorbed urokinase precursor was eluted with a 0.2 M aqueous glycine-HCl solution (pH: 2.5) containing 0.5 M NaCl. The elute was neutralized and then was passed through a carrier fixed anti-mouse IgG rabbit IgG and was removed a very small amount of exuded mouse IgG. The solution thus passed was freed from bacteria and filtered, and then freeze dried to obtain a highly purified urokinase precursor having a * Tr3de M3rk 1 3~6585 1 specific activity of at least 80,000 IU/mg.
This purified product, when measured with SDS
polyacrylamide gel electrophoresis, showed one band ascribable to a molecular weight of 50,000.
While the invention has been described in detail and with reference to specific embodiment thereof, it will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope thereof.
An object of the present invention is to provide a method of stabilizing a urokinase precursor and also to a dry preparation containing a stabilized urokinase precursor.
It has been found that a dried urokinase precursor is stabilized when a mixture of (a) at least one member of inorganic salts, organic acid salts, and polar amino acids and their salts, and (b) human albumin is added to the precursor.
In one embodiment, the present invention relates to a method of stabilizing a urokinase precursor by adding thereto (a) at least one member of inorganic salts, organic acid salts, and polar amino acids and their salts, and (b) albumin.
In another embodiment, the present invention relates to a dry preparation containing a stabilized urokinase precursor.
DETAILED DESCRIPTION OF THE INVENTION
The urokinase precursor as used herein includes those precursors obtained from urine, human kidney cells, and by genetic engineering (see JP-A-62981/1985 corresponding to Canadian Patent Application Serial 1 3365~5 1 No. 462,860 for example).
The effect of the present invention is particularly remarkable in a highly purified urokinase precursor, such as a urokinase precursor of a concentration that the specific activity is 100,000 IU/mg protein. The symbol "IU" is an abbreviation of an international unit of UK. 1 IU/ml, which is e~uivalent to the UK activity when 1 ml of the precursor is treated with plasmin, means that 1 ml of the precursor, when treated with plasmin, has the same activity as that of 1 IU of UK.
Examples of the inorganic salts that are used in the present invention are alkali metal and alkaline earth metal salts of halogen, and alkali metal and alkaline earth metal salts of phosphoric acid. Preferred are sodium chloride, potassium chloride, sodium phosphate, potassium phosphate, and calcium phosphate.
As the organic acids, aliphatic carboxylic acids which may have a hydroxyl group are preferably used. The number of carboxyl groups is preferably 1 to 3 and the number of hydroxyl groups is preferably 0 to 3. Suitable examples of these organic acids are oxy acids such as citric acid, aliphatic dicarboxylic acids such as oxalic acid, and fatty acids such as acetic acid and mandelic acid.
-4- l 336585 l Examples of the salts of the above organic acids are alkali metal (e.g., sodium and potassium) salts.
Examples of the polar amino acids that are used in the present invention are glutamic acid, aspartic acid, arginine, lysine, and histidine.
Suitable examples of the salts of the above polar amino acids are sodium glutamate and arginine chloride.
Among the above substances which can be used as component (a), organic acid salts and poler amino acids, more particularly arginine and sodium citrate, are preferred.
The albumin as used herein is preferably albumin obtained from human in view of the problem of antigenicity. There are no special limitations to the albumin as long as it is purified for the purpose of use for medical treatment. The purity is preferred to be such that the albumin content as determined by electrophoresis is not less than 80%. Methods of obtaining human albumin include the ethanol fractionation method (see Japanese Patent Publication Nos. 2869/72 and 5297/60), and the method comprising heating in the presence of organic acids (see Japanese Patent Publication Nos. 1604/68 and 40132/76). It is particularly preferred to subject the albumin to heat treatment (preferably at 60C for about 10 hours) for inactivation of hepatitis virus, for example.
-_ 5 _ t 33 65~5 l Albumin is added in an amount corresponding to 10 to 60mg, preferably 25 to 50 mg, and the inorganic salt, organic acid salt, or polar amino acid or its salt is added in an amount corresponding to 2 to 50 mg, preferably 5 to 30 mg, per 10,000 to 250, 000 IU of the urokinase precursor.
The stabilizers of the present invention, i.e., inorganic salts, organic acid salts, polar amino acids or their salts, are added with good results at any stage during the process that the urokinase precursor is placed under conditions where the precursor could be inactivated, such as the purification and storage of the urokinase precursor.
The present invention is described in greater detail with reference to the following examples.
The activity of the urokinase precursor was measured, after activation with plasmin, by the use of a synthetic substrate (Glt-Gly-Arg-MCA).
To a solution of a purified urokinase precursor (specific activity: 135,000 IU/mg protein) in a 0.10 M
phosphate buffer (pH:6.0) as a solvent was added a solution a solution of albumin in the same buffer as above to prepare 25,000 IU/ml of an aqueous urokinase precursor solution containing 20 mg/ml of human albumin. Various additives (inorganic salts, organic acid salts, or polar - 6 ~ t 3 3 6 5 ~ 5 1 amino acids or their salts) were added to the above prepared aqueous urokinase precursor solution so that the total amount of the additives added was 8 mg/ml and then freeze dried. For comparison, the aqueous urokinase precursor solution not containing any additive, and the aqueous urokinase precursor solution with only human albumin added thereto were freeze dried in the same manners as above.
These compositions were measured for the remaining urokinase precursor titer (%) just after its freeze drying and after storage at 50C for 3 months. The results are shown in Table 1.
1 Table 1 Residual Titer (~) After Storage Run Human Additive Just after at 50C for No. Albumin (Total Amount) Freeze Dryinq 3 Months (mg/ml) 1 20 Sodium citrate 99.5 99.8 - (8 mg/ml) 2 " Sodium mandelate 99.0 96~1 (8 mg/ml) 3 " Sodium phosophate 96.5 86.5 (8 mg/ml) 4 " NaCl (Table salt) 98.6 95.2 (8 mg/ml) n Potassium chloride 96.5 86.2 (8 mg/ml) 6 " Aspartic acid 97.5 93.8 (6.4 mg/ml) 7 " Glutamic acid 97.2 94.2 (6.4 mg/ml) 8 " Sodium glutamate 97.6 95.8 (6.4 mg/ml) g n Arginine 99.5 98.8 (6.4 mg/ml) n Lysille (6.4 mg/ml) 11 n His~idine 96.5 - 88.6 (6.4 mg/ml) 12 " - 96.7 -- 60.2 3013 - - 94.0 2.5 Note: Run Nos. 1 - 11: Examples of the present invention Run Nos. 12 and 13: Control Freeze dried preparations were prepared in the same manner as in Experiment 1 containing human albumin, sodium citrate, salt (NaCl), arginine, or sodium glutamate which rates were varied.
The preparations were measured for the residual urokinase precursor titer (%) just after preparation and after storage at 50C for 1 month. The results are shown in Table 2.
Table 2 Formation Residual Titer (%) Additive Just After After Storage Run Human Amount Freezeat 50C for No. Albumin Type (mq/ml) Dryinq1 Month 1 20 Sodium citrate - 77.1 75.2 mg/ml - 2 ~ do 3.2 87.3 86~4 3 ~ do 6.4 99.3 99.2 4 Salt - 76.3 75.2 " do 3.2 84.2 84.1 6 - do 6.4~ 97.8 97.4 7 Arginine - 94.0 75.2 8 ~ do 3.2 99.5 86.4 9 ~ do 6.4 99.5 99.2 . Sodium glutamate - 94.0 75.2 11 i~ do 3.2 97.0 84.1 12 n do 6.4 97.6 97.4 l The titer shown in Tables 1 and 2 is a residual activity against the titer before the above treatment as 100 .
25,000 IU of a purified urokinase precursor (specific activity: 135,000 IU/mg protein), 20 mg of human albumin, and 6.4 mg of sodium citrate were dissolved in 1 ml of a 0.1 M phosphate buffer (pH, 7.0), aseptically filtered, charged in a vial bottle, and then freeze dried to prepare 10 an injection preparation containing 25,000 units of urokinase precursor, 20 mg of human albumin and 6.4 mg of sodium citrate.
A preparation containing 25,000 IU of urokinase l5 precursor, 20mg of human albumin and 6.4 mg if table salt (NaCl) was prepared in the same manner as in Example 1.
So,OoO IU of a purified urokinase precursor (specific activity: 135,000 IU/mg protein), 40 mg of human 20 albumin and 12.8 mg of sodium citrate were processed in the same manner as in example 1 to prepare a preparation containing 50,000 IU of urokinase precursor, 40 mg of human albumin and 12.8 mg of sodium citrate.
- lo - 1 3 3 6 5 8 5 25,000 IU of a purified urokinase precursor (specific activity: 135,000 IU/mg protein), 20 ~ of human albumin, and 6.4 mg of arginine were dissolved in 1 ml of a O.lM phosphate buffer (pH, 7.0), aseptically filtered, charged in a vial bottle, and then freeze dried to prepare a preparation containing 25,000 IU of urokinase precursor, 20 mg Of human albumin and 6.4 mg of arginine.
A preparation containing 25,000 IU of urokinase precursor, 20 mg of human albumin and 6.4 mg of sodium glutamate was prepared in the same manner as in Example 1.
50,000 IU of a purified urokinase precursor (specific activity: 135,000 IU/mg Protein), 40 mg of human albumin and 12.8 mg of arginine were processed in the same manner as in Example 1 to prepare a preparation containing 50,000 IU of urokinase precursor, 40 mg of human albumin and 12.8 mg of arginine.
SYNTHESIS EXAMPLE
Cultivated human kidney cells were grown for 3 days in a 0.1% human serum albumin-added non-serum culture, and the fermentation broth thus obtained was subjected to centrifugal separation. The resulting supernatant was frozen and stored. The pooled supernatant was adjusted to l pH 5.5 and contacted with CM-Sephadex C-50. After washing a column with a 0.16 M phosphate buffer (pH 5.5), the adsorbed urokinase precursor was eluted with a 0.16 M phosphate buffer (pH, 8.5).
Among hybridomas prepared by fusion using polyethylene glycol of spleen cells of BALB/c mouse which had been immunized with the urokinase precursor and mouse myeloma cells, a clone having a high productivity of antibody for the urokinase precursor was selected. From this fermentation broth of the fused cells, an anti-urokinase monoclonal antibody was recovered. This monoclonal antibody was immobilized on BrCN-activated Sepharose 4B (produced by Pharmacia Co., Ltd.) This monoclonal antibody column was equlibrated with a 0.1 M phosphate buffer (pH, 7.0) containing 0.4 M
NaCl, and then contacted with the elute containing urokinase precursor. After washing the column with a 0.1 M phosphate buffer (pH, 7.0) containing 0.4 M NaCl, the adsorbed urokinase precursor was eluted with a 0.2 M aqueous glycine-HCl solution (pH: 2.5) containing 0.5 M NaCl. The elute was neutralized and then was passed through a carrier fixed anti-mouse IgG rabbit IgG and was removed a very small amount of exuded mouse IgG. The solution thus passed was freed from bacteria and filtered, and then freeze dried to obtain a highly purified urokinase precursor having a * Tr3de M3rk 1 3~6585 1 specific activity of at least 80,000 IU/mg.
This purified product, when measured with SDS
polyacrylamide gel electrophoresis, showed one band ascribable to a molecular weight of 50,000.
While the invention has been described in detail and with reference to specific embodiment thereof, it will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope thereof.
Claims (18)
1. A method of stabilizing a urokinase precursor which comprises adding (a) at least one member selected from the group consisting of inorganic salts, organic acid salts, and polar amino acids and salts thereof, and (b) human albumin to the urokinase precursor, wherein said albumin is added in an amount of 10 to 60 mg per 10,000 to 250,000 IU
of the urokinase precursor, and said inorganic salt, organic acid salt, or polar amino acid or salt thereof is added in an amount of 2 to 50 mg per 10,000 to 250,000 IU of the urokinase precursor.
of the urokinase precursor, and said inorganic salt, organic acid salt, or polar amino acid or salt thereof is added in an amount of 2 to 50 mg per 10,000 to 250,000 IU of the urokinase precursor.
2. The method as claimed in Claim 1, wherein said inorganic salt is selected from the group consisting of sodium chloride, potassium chloride, sodium phosphate, potassium phosphate and calcium phosphate.
3. The method as claimed in Claim 1, wherein said organic acid is selected from the group consisting of citric acid, oxalic acid, acetic acid and mandelic acid.
4. The method as claimed in Claim 1, wherein said polar amino acid and salts thereof are glutamic acid, aspartic acid, arginine, lysine, histidine and salts thereof.
5. The method as claimed in Claim 1,2,3 or 4, wherein said albumin is subjected to heat treatment at 60°C
for about 10 hours prior to the addition the urokinase precursor.
for about 10 hours prior to the addition the urokinase precursor.
6. A urokinase precursor-containing dry preparation comprising a urokinase precursor as a major component and further, as stabilizers, (a) at least one member selected from the group consisting of inorganic salts, organic acid salts, and polar amino acids and salts thereof, and (b) human albumin, wherein said albumin is added in an amount of from 10 - 60 mg/10,000 to 250,000 IU
of the urokinase precursor, and said inorganic salt, organic acid salt or polar amino acid or salt thereof is added in an amount of from 2 to 50 mg/10,000 to 250,000 IU of the urokinase precursor.
of the urokinase precursor, and said inorganic salt, organic acid salt or polar amino acid or salt thereof is added in an amount of from 2 to 50 mg/10,000 to 250,000 IU of the urokinase precursor.
7. The preparation as claimed in Claim 6, wherein the polar amino acid and salts thereof are glutamic acid, aspartic acid, arginine, lysine and histidine, and salts thereof.
8. The preparation as claimed in Claim 6, wherein said inorganic salt is selected from the group consisting of sodium chloride, potassium chloride, sodium phosphate, potassium phosphate and calcium phosphate.
9. The preparation as claimed in Claim 6, wherein said organic acid is selected from the group consistingof citric acid, oxalic acid, acetic acid and mandelic acid.
10. The preparation as claimed in Claim 6, 7, 8 or 9, wherein said albumin is a heat treated albumin treated at 60°C for about 10 hours.
11. A method of stabilizing a urokinase precursor which comprises adding (a) at least one member selected from the group consisting of inorganic salts and organic acid salts other than polar amino acid salts, and (b) human albumin to the urokinase precursor, wherein said albumin is added in an amount of 10 to 60 mg per 10,000 to 250,000 IU
of the urokinase precursor, and said inorganic salt or organic acid salt is added in an amount of 2 to 50 mg per 10,000 to 250,000 IU of the urokinase precursor.
of the urokinase precursor, and said inorganic salt or organic acid salt is added in an amount of 2 to 50 mg per 10,000 to 250,000 IU of the urokinase precursor.
12. The method as claimed in Claim 11, wherein said inorganic salt is selected from the group consisting of sodium chloride, potassium chloride, sodium phosphate, potassium phosphate and calcium phosphate.
13. The method as claimed in Claim 11, wherein said organic acid is selected from the group consisting of citric acid, oxalic acid, acetic acid and mandelic acid.
14. The method as claimed in Claim 11, 12 or 13, wherein said albumin is subjected to heat treatment at 60°C
for about 10 hours prior to the addition to the urokinase precursor.
for about 10 hours prior to the addition to the urokinase precursor.
15. A urokinase precursor-containing dry preparation comprising a urokinase precursor as a major component and further, as stabilizers, (a) at least one member selected from the group consisting of inorganic salts and organic acid salts other than polar amino acid salts, and (b) human albumin wherein said albumin is added in an amount of from 10-60 mg/10,000 to 250,000 IU of the urokinase precursor, and said inorganic salt or organic acid salt is added in an amount of from 2 to 50 mg/10,000 to 250,000 IU of the urokinase precursor.
16. The preparation as claimed in Claim 15, wherein said inorganic salt is selected from the group consisting of sodium chloride, potassium chloride, sodium phosphate, potassium phosphate and calcium phosphate.
17. The preparation as claimed in Claim 15, wherein said organic acid is selected from the group consisting of citric acid, oxalic acid, acetic acid and manelic acid.
18. The preparation as claimed in Claim 15, 16 or 17, wherein said albumin is a heat treated albumin treated at 60°C for about 10 hours.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP60079428A JPS61238731A (en) | 1985-04-16 | 1985-04-16 | Method of stabilizing urokinase precursor and dried preparation |
| JP79429/85 | 1985-04-16 | ||
| JP79428/85 | 1985-04-16 | ||
| JP60079429A JPS61238732A (en) | 1985-04-16 | 1985-04-16 | Method of stabilizing urokinase precursor and dried preparation |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1336585C true CA1336585C (en) | 1995-08-08 |
Family
ID=26420450
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000506647A Expired - Fee Related CA1336585C (en) | 1985-04-16 | 1986-04-15 | Method of stabilizing urokinase precursor and dry preparation containing said precursor |
Country Status (5)
| Country | Link |
|---|---|
| EP (1) | EP0200966B1 (en) |
| KR (1) | KR940003056B1 (en) |
| CA (1) | CA1336585C (en) |
| DE (1) | DE3688713T2 (en) |
| ES (1) | ES8706821A1 (en) |
Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS61176532A (en) * | 1985-01-30 | 1986-08-08 | Green Cross Corp:The | Stablization of plasminogen activator precursor |
| JP2545231B2 (en) * | 1987-06-18 | 1996-10-16 | 日本ケミカルリサ−チ株式会社 | Method for suppressing molecular weight reduction of urokinase by heating |
| CA2007901A1 (en) * | 1989-01-27 | 1990-07-27 | Hiroshi Matsuda | Urokinase precursor-lipid composite |
| JPH02268681A (en) * | 1989-04-07 | 1990-11-02 | Green Cross Corp:The | Stabilization of urokinase precursor and dried preparation |
| EP0395918A3 (en) * | 1989-04-13 | 1991-10-23 | Vascular Laboratory, Inc. | Plasminogen activator complex of pure pro-urokinase covalently bound by a disulfide bridge to human serum albumin |
| US8545459B2 (en) | 2009-02-25 | 2013-10-01 | Teleflex Medical Incorporated | Stabilized enzyme compositions |
| US9333280B2 (en) | 2009-02-25 | 2016-05-10 | Teleflex Medical Incorporated | Stabilized enzyme compositions |
| EP3052051B1 (en) | 2013-09-30 | 2019-06-12 | Teleflex Medical Incorporated | Stabilized enzyme compositions |
Family Cites Families (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4245051A (en) * | 1978-03-30 | 1981-01-13 | Rockefeller University | Human serum plasminogen activator |
| JPS54147916A (en) * | 1978-05-12 | 1979-11-19 | Sumitomo Chem Co Ltd | Preparation of urokinase injection |
| JPS58224687A (en) * | 1982-06-24 | 1983-12-27 | Toubishi Yakuhin Kogyo Kk | Plasminogen-activation enzyme agent and novel process for preparation thereof |
| JPS59139323A (en) * | 1983-01-28 | 1984-08-10 | Green Cross Corp:The | Dried urokinase preparation |
| EP0139447B1 (en) * | 1983-09-13 | 1992-12-30 | The Green Cross Corporation | A process for preparing urokinase zymogen |
| EP0151996B1 (en) * | 1984-01-30 | 1991-04-03 | Asahi Kasei Kogyo Kabushiki Kaisha | Process for the preparation of a double-chain plasminogen activator |
| JPS61176532A (en) * | 1985-01-30 | 1986-08-08 | Green Cross Corp:The | Stablization of plasminogen activator precursor |
-
1986
- 1986-04-15 KR KR1019860002879A patent/KR940003056B1/en not_active IP Right Cessation
- 1986-04-15 CA CA000506647A patent/CA1336585C/en not_active Expired - Fee Related
- 1986-04-16 ES ES554477A patent/ES8706821A1/en not_active Expired
- 1986-04-16 EP EP86105235A patent/EP0200966B1/en not_active Expired - Lifetime
- 1986-04-16 DE DE86105235T patent/DE3688713T2/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| DE3688713D1 (en) | 1993-08-26 |
| EP0200966A3 (en) | 1988-09-28 |
| ES8706821A1 (en) | 1987-07-01 |
| KR860008199A (en) | 1986-11-12 |
| KR940003056B1 (en) | 1994-04-13 |
| ES554477A0 (en) | 1987-07-01 |
| EP0200966A2 (en) | 1986-11-12 |
| DE3688713T2 (en) | 1993-12-16 |
| EP0200966B1 (en) | 1993-07-21 |
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